Five broad, long term goals of this laboratory are (i) to increase understanding of the molecular determinants of substrate specificity of (chymo) trypsin-like serine proteases, a large gene family that includes many medically important members and is among the most advanced model systems of enzymology, (ii) to develop powerful and robust techniques to distinguish the specificities of very closely related proteases, (iii) to facilitate the rational design of highly specific inhibitors of individual proteases, which can be used to examine the role of a particular protease in complex biological or pathological processes, (iv) to aid the de novo design of novel proteases with desired substrate specificities, and (v) to assist the development of novel therapeutic agents for the treatment of acute myocardial infarction and other thromboembolic disorders. The specific aims of this renewal application are: 1 ) To elucidate optimal subsite occupancy for substrates of all three proteases of the human fibrinolytic cascade, tissue type plasminogen activator (t-PA), urokinase (u-PA), and plasmin, using both conventional and novel substrate phage display protocols. 2) To use site specific mutagenesis to identify and characterize molecular determinants of the stringent substrate specificities of t-PA and u-PA. 3) To design and characterize high affinity, specific inhibitors of u- PA. The proposed studies are interdisciplinary and involve the use of techniques of molecular biology (e.g., site directed mutagenesis, phage display), biochemistry (enzyme expression and purification, enzyme and inhibitor kinetics), biophysics (molecular modeling, X-ray crystallography), and chemistry (synthesis and characterization of small peptide substrates and peptidic inhibitors of serine proteases). The primary objective of this proposal, to increase understanding of the activity, specificity, and selective inhibition of fibrinolytic serine proteases, ensures that these studies will be directly relevant to human health. Administration of one of these enzymes (t-PA) is currently the standard treatment for acute myocardial infarction, the major killer of both American men and women. The development of novel plasminogen activators with altered or new properties, therefore, may provide improved thrombolytic agents. In addition, the development of high affinity, highly selective inhibitors of u-PA may provide new therapeutic tools to combat restenosis following invasive vascular procedures such as angioplasty.